Proteolytic enzyme

ABSTRACT

The invention relates to an extremely thermophilic bacterium, Thermus T-351, and a thermophilic protease, Caldolysin, derived therefrom. The bacterium cells are Gram negative, non-sporulating rods. Its natural environment is a hot pool at 79°±4° C. The protease is stable at temperatures up to 75° C. at a pH range of 4 to 12. It is most active at temperatures of 65° C. to 85° C. but retains at least some activity at lower temperatures.

FIELD OF THE INVENTION

This invention relates to a novel micro-organism capable of producing athermostable extracellular proteolytic enzyme. It also relates to themethod of preparing the proteolytic enzyme and to the proteolytic enzymeitself.

SUMMARY OF THE INVENTION

It is recognised that there is a demand for thermostable proteolyticenzymes in the food, fermentation, animal feed and pharmaceuticalindustries which is not being entirely met. The inventors have succeededin isolating a micro-organism from a hot pool in the thermal area ofRotorua, New Zealand, which organism is capable of producing athermostable proteolytic enzyme.

It is an object of this invention to provide starting material capableof producing a thermostable proteolytic enzyme and to provide aproteolytic enzyme which goes some way towards meeting theaforementioned demand or at least provides the public with a usefulchoice.

Accordingly the invention may be said broadly to consist in ThermusT-351 (as herein defined) in a substantially biologically pure form.

In another aspect the invention may be said broadly to consist in aprocess for isolating Thermus T-351 which comprises isolating a sampleof solution from a hot pool (at 79°±4° C.) low in sulphide at pH 7.8containing said micro-organism, preparing a culture from said sample andmaintaining said culture at a temperature of 65° C. to 85° C. and a pHbetween 7.2 and 8.2 for a time sufficient to produce an adequate yieldand isolating the Thermus T-351 from said culture during the late logphase thereof.

In another aspect the invention may be said broadly to consist inThermus T-351 whenever prepared by the foregoing process.

In another aspect the invention may be said to consist in Caldolysin (asherein defined) in a substantially pure form.

In another aspect the invention may be said broadly to consist in amethod of preparing Caldolysin (as herein defined) which comprisescentrifuging a culture of Thermus T-351, subjecting the supernatant fromsaid centrifugation to affinity chromatography on a suitable activatedaffinity chromatography gel and eluting the Caldolysin from said gelwith a suitable buffer.

In another aspect the invention may be said to consist in Caldolysin (asherein defined) whenever prepared by the process hereinbefore defined.

In another aspect the invention may be said broadly to consist inCaldolysin (as herein defined) immobilised on a suitable substrate.

DETAILED DESCRIPTION OF THE INVENTION

The invention consists in the foregoing and also envisages constructionsof which the following gives examples.

DEFINITIONS

1. Thermus T-351

This micro-organism was isolated from Hot Pool No. 351 as marked on themap of "Whakarewarewa Hot Springs", 1:2000, 1st Edition by New ZealandGeological Survey (Bibliographic reference--Lloyd, E. F. 1974, Geologyof Whakarewarewa hot springs, DSIR information series No. 104 DSIR,Wellington, N.Z.). The hot pool was at (79°±4° C.), was low in sulphide,at pH 7.5 to 7.8. It grew poorly below 60° C. It was obligately aerobic.The cells were Gram negative, non-motile, non-sporulating rods. It issimilar to Thermus aquaticus (Brock et al., J. Bacteriol 98, 289-287;Degryse et al, Archives of Microbiology 117, 18) but the inventors havenoted a significant difference in cytochrome composition of betweenThermus-351 and Thermus aquaticus. The product exhibits optimal activityat 70° to 80° C. and negligible activity below 40° C. Other propertiesof this microorganism are set out herein below and are also described inHickey and Daniel, J. of Gen. Microbiology (1979), 114, 195-200, thetext of which is hereby incorporated by reference.

2. Caldolysin

This is a proteolytic enzyme produced by Thermus T-351 and isolated bythe process described below. It is a protease having molecular weight20,000±3,000. It has an isoelectric point of approximately 8.5 and itsenzymatic activity is described below. This enzyme is stable attemperatures of 75° C. and below, in the presence of divalent cations,particularly calcium ions. It is stable at pH values from 4 to 12 in thepresence of calcium ions. Other properties and the method of preparationof this product are set out herein below.

The invention will now be described in more detail with reference to theaccompanying drawings, in which

FIG. 1 is a flow scheme illustrating a two-step Caldolysin purification;and

FIG. 2 is a flow scheme illustrating a multi-step Caldolysinpurification.

EXAMPLE 1a Isolation of Thermus T-351

Samples were taken from the hot pool identified under the definition ofThermus T-351 herein above. Isolation was carried out by repeatedsub-culturing at 75° C. of the organisms contained in a 1 ml sample fromthe hot pool, in 10 ml of half strength nutrient broth, pH 8.0: This wasfollowed by growth in the medium described in example 1b (i).

EXAMPLE 1b Cultivation of Thermus T-351

(i) Cultures were maintained on a medium consisting of Allen's salts,(Jackson et al., Archiv. f/u/ r Mikrobiol 88, 127-133), with 0.1% w/vyeast extract (BBL) and 0.1% w/v trypticase (BBL) in liquid culture at75° C. The medium was adjusted to pH 8.2 prior to autoclaving. The finalpH was 7.5.

(ii) The organism was grown at 75° C. on a similar medium but with 0.3%yeast extract and 0.3% trypticase. 500 ml batches were grown in 2 lErlenmeyer flasks in an orbital incubator, and either harvested for use,or used to inoculate a 20 l fermentor. The organism grow well at 75° C.under the conditions (i) and (ii) and poorly below 60° C. Cells wereharvested during late log phase (10-12 hours after inoculation) at anabsorbance of about 1.4 at 650 nm (about 2.5×10⁷ cells ml⁻¹)

1c. Preparation of subcellular fractions:

Cell fractions were prepared as described by Daniel (Biochem et BiophysActa 216, 328-341) except that the sedimentation of small particles wascarried out at 250,000 g for 1 hour.

1d. Measurement of oxidase system activities:

Oxygen uptake was measured polarographically using a Rank electrode(Rank Brothers, Bottisham, Cambridge, England). The reaction mixtureconsisted of 0.1M KH₂ PO₄ /Na₂ HPO₄ buffer pH 7.0, a suitable amount ofmembrane particle protein, 50 μmol of substrate (except in the case ofNADH were 5 μmol were used), in a final volume of 2.5 ml.

Buffers were equilibrated at the desired temperature with sparged airfor 30 minutes.

Particles were equilibrated in the electrode for 2 minutes prior tomeasurement of oxygen uptake. Rates were measured over the first minute.

1e. Spectrophotometry:

Difference spectra were obtained at room temperature with a Cary model17 recording spectrophotometer.

The concentration of individual cytochromes was determined from thedithionite-reduced minus oxidised difference spectra, and for cytochromeo from the reduced minus reduced + CO difference spectra, using thefollowing wavelength pairs and extinction coefficients: c-typecytochrome, 553-540, ε_(mM) =19 mM⁻¹ cm⁻¹ (Chance et al J. Bio. Chem.217 439-451) total b-type cytochrome, 569-575 nm, ε_(mM) =17.5 mM⁻¹ cm⁻¹(Deeb & Hanger (J. Bio. Chem. 239, 1024-1031); cytochrome o-CO, 417-429,ε_(mM) =170 mM⁻¹ cm⁻¹ (Daniel, (Biochim et Biophys. Acta 216, 328-341);a-type cytochrome, 602-630 and 613-630 ε_(mM) =24 mM⁻¹ cm⁻¹ (Van Galder,(Biochim. et Biophys. Acta 118, 36-46).

Washed membrane particles were able to oxidise other substratesincluding glutamate and malate (>0.05 μmol 0₂ min⁻¹ (mg protein)⁻¹), andlactate, citrate, fumerate, glycerol and glucose (0.005-0.02 μmol 0₂min⁻¹ (mg protein)⁻¹). Except in the case of succinate and lactate,activities were enhanced by added supernatant and by NADH. Acetate,sucrose, mannitol and ethanol were not oxidised.

Both NADH and succinate oxidases had maximum activity at pH 7.0, and ata phosphate buffer molarity of 0.1M, as determined at 75° C.

The activities of NADH and succinate oxidases were determined after 2minutes preincubation at temperatures between 40° C. and 95° C. Each washighest at 75° C. in cell free extract and in large and small particles.The NADH oxidase rate in respiratory particles was particularlytemperature sensitive, the rates at 70° C. and 80° C. being about halfthat at 75° C. In all cases activity at 75° C. was at least 10-foldgreater than that at 40° C.

At 75° C., apart from an initial partial loss of activity therespiratory chain in whole cells, cell free extracts and respiratoryparticles was relatively stable, but there was a substantial short termincrease in succinate respiration of whole cells and endogenousrespiration followed a similar pattern. At 90° C. this was found forwhole cells and cell free extracts, but not washed respiratoryparticles. At 90° C. the succinate oxidase of whole cells and the NADHoxides of washed respiratory particles were substantially less stablethan the oxidase activities of cell free extract.

These stabilities are appreciably greater than those reported for NADHoxadase from Bacillus stearothermophilus protoplasts (Wisdom & Walker,(J. Bacteriol, 114 1336-1345).

The thermostability of the NADH oxidase activity of respiratoryparticles at 90° C. over a 15 minute period was unaffected by phosphatebuffer concentration (0.01M to 2.0M), 1.0M-MgSO₄ or by 10 mg ml⁻¹casein. Stability was enhanced about 2-fold by 50% (v/v) glycerol,2.0M-(NH₄)₂ SO₄, and 10 mg ml⁻¹ NADH. Rates were determined at 75° C.

Absorption peaks of a, b, and c-type cytochromes in washed respiratoryparticles at 613 and 602, 559 and 555 nm respectively were recorded. Themajor a-type cytochrome had an absorption peak at 613 nm, which isunusual: the troughs at 615 and 444 nm in the carbon monoxide spectrasuggest that at least one of the a-type cytochromes is a terminaloxidase. The trough at 561 nm and the peak at 417 nm indicate thepresence of cytochrome o, and the trough at 550 nm suggests that therewas some CO-reactive c-type cytochrome in the respiratory particles. Thehigh speed supernatant contained at least two soluble c-type cytochromessince the ratio of the peaks at 420 and 426 nm varies somewhat betweenpreparations, and at least one of these was CO-reactive.

b and c-type cytochromes in the Thermus NH have been reported byPask-Hughes & Williams (Scientific Progress at Oxford 62, 373-393) anda-605 and b and c-type cytochromes in a Thermus aquaticus type organismby McFetters and Ulrich (J. Bacterial 110(2), 777-779).

Cytochrome concentrations (μmol cytochrome (g. pro-tein)⁻¹) inrespiratory particles were a-602, 0.03; a-613, 0.06; total b-type, 0.89;o-0.21; total c-type 0.64: In the supernatant, c-type 0.79; CO-reactivecytochrome c, 0.02. These concentrations are fairly typical of thesefound in other aeorobes.

All inhibitors tested produced levels of inhibition within the range ofthose found in other bacteria and there was no evidence that activesites were less exposed than in non-thermophiles. Terminal oxidaseinhibitors affected NADH and succinate oxidases equally, as did amytal.Rotenone had more effect on the NADH oxidase, while Bathophenanthroline2-heptyl-4-hydroxyquinoline-N-oxide and antimycin A were all moreeffective inhibitors of succinate oxidase.

EXAMPLE 2a Two Step Caldolysin Purification

The culture fluid treated according to the flow scheme illustrated inFIG. 1 comes from Example 1b. The centrifugation is conducted in acontinuous flow centrifuge at 27,000 g. The pH of the Supernatant wasadjusted to pH 8 prior to its being passed through the affinity gel.

                                      TABLE 1                                     __________________________________________________________________________                             Specific                                                       Volume                                                                             [Protein]                                                                          Activity                                                                           Activity                                                                           Purification                                                                        Yield                                     STEP      (1.) (μg/ml)                                                                         (PU/mg)                                                                            (PU/ml)                                                                            (fold)                                                                              (%)                                       __________________________________________________________________________    1. Supernatant                                                                          20   24   .005  .25  1.0  100                                       2. Affinity Purified                                                                    1.1  14   .079 5.76 23.2   73                                       __________________________________________________________________________

The experimental data set out in Table 1 herein below.

EXAMPLE 2b Multistep Caldolysin Purification

The overall reaction scheme is illustrated in FIG. 2, and the majorityof the details are set out in FIG. 2. However the ultrafiltration stepconcentrated the eluate (2) ten times. The retentate (3) subjected toaffinity chromatography was adjusted to pH 8. the absorbate 4 was elutedas a single peak with pH 2.7 0.1M acetic acid containing 10 mM Ca²⁺.

The enzyme concentrate (5) was eluted from the G75 gel column using aneluting buffer at pH 8.1 and 10 mM Ca²⁺.

The data of the various steps in the reaction scheme are set out hereinbelow in table 2.

                                      TABLE 2                                     __________________________________________________________________________                         Total activity                                                            Total                                                                             Proteolytic                                                               protein                                                                           Units* Sp. Act                                                                           Yield                                         Step        Vol. (L)                                                                           (mg)                                                                              (× 1000)                                                                       PU/mg                                                                             %   Purification                              __________________________________________________________________________    1. Supernatant                                                                            50   4000                                                                              5.9    1.23                                                                              100 1.0                                       2. SPC25 Eluate                                                                           49.5 2740                                                                              4.03   1.47                                                                              68.3                                                                              1.2                                       3. Millipore concentrate                                                                  8    1410                                                                              3.47   2.46                                                                              58.8                                                                              2.0                                       4. Affinity purified enz.                                                                 2.5   36 1.67   46.4                                                                              28.3                                                                              37.7                                      5. Lyophilisate conc.                                                                     0.3   36 1.23   34.2                                                                              20.8                                                                              27.8                                      6. G75 purified enz.                                                                      1.5  8.75                                                                              0.97   110.9                                                                             16.4                                                                              9.2                                                                           (fold)                                    __________________________________________________________________________     *1 PU = mg tyrosine/min at 75° C., substrate 0.5% casein               (Hammersten)                                                             

EXAMPLE 3 Properties of Caldolysin A. Physical

(1) A Molecular weight of 20,000±3,000 was determined by gelchromatography, SDS electrophoresis, and Gradipore electrophoresis.

(2) Isoelectric point: 8.5±0.5.

(3) Response to inhibitors (Table 3) and enzymatic specificity indicatethat Caldolysin can be categorised as a metal-chelator-sensitive lyticprotease (see Morihara (1974): "Comparative Specificity of MicrobialProteases", Advances in Enzymology 41, 179).

Enzymatic

Caldolysin hydrolyses a range of high molecular weight proteinsubstrates (Table 4) and some low molecular weight peptide substrates(Table 5). However, a number of common peptide analogues (proteasesubstrates) are not hydrolysed (Table 5).

Caldolysin lyses a broad range of Gram-negative bacteria, but fewgram-positive microorganisms (Table 6).

C. Stability

(1) Thermostability.

In the presence of 10 mM Ca²⁺, 100% activity is retained at temperaturesof 75° C. and below for an extended period (no loss over 170 hours).Removal of Ca²⁺ markedly reduces thermostability. Half-life data attemperatures between 75° C. and 95° C. are shown in Table 4, togetherwith published data on other thermophilic proteases.

(2) pH/Stability

Caldolysin is stable (in the presence of calcium ions at 20° C.) forprotracted periods at pH values of 4.0 to 12.0. At pH 3.0 T^(1/2) =2hours.

At high and low pH values (for example pH 4 and pH 10), incubation atelevated temperatures results in a marked reduction in stability.

                  TABLE 3                                                         ______________________________________                                        INHIBITORS                                                                                                         % In-                                                                         hibitor                                  Type                                 of Ac-                                   of Action                                                                             Inhibitor      Concentration tivity                                   ______________________________________                                        General EDTA           12.5     mM     100%                                   Metal   EDTA           10       mM     70%                                    Chelator                                                                              EDTA           1        mM     40%                                            EDTA           0.13     mM     --                                     Cysteine-                                                                             Iodoacetic acid                                                                              10       mM     60%                                    Enzyme  "              2        mM     --                                     Inhibitor                                                                             "              0.25     mM     --                                     Serine- Phenylmethyl   10       mM     10%                                    Enzyme  Sulfonyl Fluoride                                                                            1        mM     --                                     Inhibitor                                                                             "              5        mM     --                                     Cysteine-                                                                             p-chloromercuri                                                                              5        mM     --                                     Enzyme  benzoate       2.5      mM     --                                     Inhibitor                                                                     Zn-specific                                                                           o-phenanthroline                                                                             10       mM     --                                     Chelator                                                                              "              1        mM     --                                     Ca-specific                                                                           EGTA           10       mM     45%                                    Chelator                                                                              EGTA           1        mM     18%                                            Trypsin inhibitor                                                                            1.0      Mg ml.sup.-1                                                                         --                                     Acid    N--α-p-tosyl-L-lysine                                                                  3 × 10.sup.-2                                                                    mM     --                                     protease                                                                              Chloromethyl ketone                                                   inhibitor                                                                             HCI                                                                   ______________________________________                                    

Although the reasons are not fully understood and we do not wish to bebound by any one theory, the apparent inhibition of Caldolysin by EDTAand EGTA is likely as the result of destabilisation caused by calciumremoval, and the subsequent loss of enzyme activity as the result ofautolysis.

                  TABLE 4                                                         ______________________________________                                        HYDROLYSIS OF PROTEINS BY CALDOLYSIN                                          Substrate    Rate of hydrolysis                                               ______________________________________                                                                   % of rate of                                                    (Δ.sub.280 min.sup.-1 × 10.sup.3)                                               casein hydrolysis                                  casein       3.33          100                                                ovalbumin    1.45          44                                                 bovine serum albumin                                                                       1.33          40                                                 haemoglobin  0.90          27                                                 collagen     0.70          21                                                 fibrin       0.65          18                                                                            % of rate of axo-                                               (Δ.sub.440 min.sup.-1 × 10.sup.3)                                               casein hydrolysis                                  azo-casein   2.75          100                                                azo-albumin  4.15          151                                                azo-collagen 0.87          32                                                              (ΔA.sub.395 min.sup.-1 × 10.sup.3)                   elastin-congo red                                                                          0.25          approx. 7                                          ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        HYDROLYSIS OF PEPTIDE AND                                                     PEPTIDE ANALOGUES BY CALDOLYSIN                                                                            Bond                                             Substrate          Hydrolysis                                                                              hydrolysed                                       ______________________________________                                        Gly-gly            -         -                                                Gly--gly--gly      -         -                                                Gly--gly--gly--gly -         gly--gly                                         Gly--gly--gly--gly--gly                                                                          -         gly--gly                                         D-leu--gly         -         -                                                L-leu--gly         -         -                                                BOC--ala--try--met--asp--                                                                        -         -                                                phe--NH.sub.2                                                                 CBZ--gly--phe--NH.sub.2                                                                          -         -                                                Acetyl-ala--ala--ala--OMe                                                                        -         ala--ala                                         CBZ--gly--pro--gly--gly--                                                                        -         gly--pro                                         pro--ala                                                                      CBZ--gly--pro--leu--gly--pro                                                                     +         pro--leu                                         Benzoyl-arginine ethyl                                                                           -         -                                                ester                                                                         CBZ--gly--p-nitro-phenyl                                                                         -         -                                                ester                                                                         Tosyl-arginine ethyl                                                                             -         -                                                ester                                                                         Benzoyl-arginine-p-                                                                              -         -                                                nitroanilide                                                                  Benzoyl-phe--val--arg--p-                                                                        +         amide                                            nitro-anilide                                                                 CBZ--gly--pro--arg--p-                                                                           -         -                                                nitroanilide                                                                  ______________________________________                                    

                                      TABLE 6                                     __________________________________________________________________________    LYSIS OF MICROORGANISMS AT 75° C. BY CALDOLYSIN                        (20 μg ml.sup.-1, 0.1 M CH.sub.3 COONa, pH 7.5)                                          ATCC Gram   Complete                                                                           Partial                                                                            No                                        Microorganism Numbers                                                                            reaction a.                                                                          lysis                                                                              lysis                                                                              lysis                                     __________________________________________________________________________    Arthrobacter globiformis                                                                    8907 +                +                                         Arthrobacter  --   +                +                                         Bacillus cereus                                                                             9373 +           +                                              Bacillus megaterium                                                                         9376 +                +                                         Bacillus circulans                                                                          9374 +           +                                              Micrococcus luteus                                                                          --   +                +                                         Micrococcus lysodeikticus                                                                   --   +           +                                              Saccharomyces cerevisiae                                                                    --   +                +                                         Sarcina lutea  196 +                +                                         Sporeformer   --   +                +                                         (unidentified Bacillus)                                                       Staphylococcus aureus                                                                       6571 +                +                                         Streptomyces griseus                                                                        8136 +           +                                              Agrobacterium tumefaciens                                                                   15955                                                                              -                +                                         Alcaligenes faecilis                                                                        8156 -      +                                                   Alcaligenes viscolactis                                                                     8154 -      +                                                   Citrobacter freundii                                                                        --   -      +                                                   Cytophaga johnsonae C.sub.4                                                                 --   -                +                                         Escherichia coli B                                                                          11303                                                                              -      +                                                   Escherichia coli K.sub.12                                                                   --   -      +                                                   Escherichia coli K.sub.12 Hfr                                                               --   -      +                                                   Escherichia coli W                                                                          --   -      +                                                   Enterobacter aerogenes                                                                      --   -      +                                                   Enterobacter cloacae                                                                        --   -      +                                                   Klebsiella pneumoniae                                                                        418 -      +                                                   Proteus vulgaris                                                                             67  -      +                                                   Pseudomonas aerogenes                                                                       --   -      +                                                   Salmonella typhimurium                                                                      --   -      +                                                   Serratia marcescens                                                                         1377 -      +                                                   Shigella flexneri                                                                           --   -      +                                                   Shigella sonnei                                                                             --   -      +                                                   __________________________________________________________________________     a. Gram reactions quoted from Bergeys Manual of Determinative Bacteriolog     (1974), 8th edition, (Buchanan R. E. and Gibbons N. E., eds.) Williams &      Wilkins Ltd.                                                             

                                      TABLE 7                                     __________________________________________________________________________    ENZYME NAME SOURCE           HALF-LIFE                                                                            TEMPERATURE                               __________________________________________________________________________    THERMOLYSIN BACILLUS         1   hr 80° C.                                         THERMOPROTEOLYTICUS                                               THERMOMYCOLASE                                                                            MALBRANCHIA PULCHELLA                                                                          1.8 hr 73° C.                             AMINOPEPTIDASE 1                                                                          BACILLUS         0.3 hr 80° C.                                         STEAROTHERMOPHILUS                                                PROTEASE    BACILLUS CALDOLYTICUS                                                                          >8.3                                                                              hr 80° C.                             CALDOLYSIN  THERMUS T-351    >24.0                                                                             hr.sup.+                                                                         75° C.                             CALDOLYSIN  THERMUS T-351    30  hr.sup.+                                                                         80° C.                             CALDOLYSIN  THERMUS T-351    5   hr.sup.+                                                                         85° C.                             CALDOLYSIN  THERMUS T-351    1   hr.sup.+                                                                         90° C.                             CALDOLYSIN  THERMUS T-351    1/2 hr.sup.+                                                                         95° C.                             __________________________________________________________________________     .sup.+ Assay conditions 0.5% casein, 75° C., 30 minutes, pH 8.1        Incubation conditions pH 8 buffer + mM Ca.sup.2+, temperature ±            0.5° C.                                                           

(3) Stabilising Effect of Divalent Cations

Table 8 shows influence of metal ions on the stability of Caldolysin at85° C. (12 μg ml⁻¹ enzyme, pH 8.1 Tris acetic acid, I=0.3M 1⁻¹).Caldolysin was dialysed in the presence of 1.0 mM EDTA to remove anymetal ion cofactors. Standard metal ion solutions were added to aliquotsof the "apoenzyme" to give 10 mM concentration, after which thethermostability of the enzyme was determined.

                  TABLE 8                                                         ______________________________________                                                             Half-life                                                       Metal ion     (minutes)                                                ______________________________________                                               Calcium       est. 340                                                        Zinc          144                                                             Strontium     155                                                             Magnesium     86                                                              Cobalt        60                                                              Barium        43                                                              Copper        21                                                              None          est. 5-10                                                ______________________________________                                    

(4) Stability to Denaturing Agents

Caldolysin has been found to be stable and active in the presence of avariety of denaturing agents as shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        Stability of CALDOLYSIN in the present of denaturing agents.                  Denaturing agent                                                                           Half life at 18° C.                                                                 Half life at 75° C.                          ______________________________________                                        1% SDS       >>13 hours   >5       hours                                      8M Urea      >>67 hours   53       minutes                                    0.8M Urea    >>13 hours   148      minutes                                    6M Guanidine HCl                                                                           >>31 hours   59       minutes                                    1% Triton X100                                                                             --           >>60     minutes                                    1% Tween 80  --           >>60     minutes                                    ______________________________________                                    

(5) Other

Caldolysin is stable to concentration by lyophilisation (freeze-drying)and rotary evaporation (reduced pressure at 37° C.) as shown in Table10.

                  TABLE 10                                                        ______________________________________                                        Method of      Concentration                                                                             % Specific                                         Concentration  Factor      Activity Loss                                      ______________________________________                                        Lyophilisation  7           7.2%                                              Rotary Evaporation                                                                           20          <5.0%                                              ______________________________________                                    

pH/Activity Relationships

Optimum pH for activity on azocasein occurs at 8.5±0.5. (at 75° C.). AtpH 6.0-pH 9.5, more than 80% of optimal activity is retained.

E Temperature/Activity Relationships

Below 40° C. enzyme activity is low (less than 6% of activity at 80°C.). Activity rises almost linearly between 45° C. and 80° C.

NB: Although % activity is low at normal temperatures (20°-40° C.),sufficient activity for effective proteolysis can be obtained simply byusing larger quantities of enzyme. However, its usefulness is clearlymaximal at 65° C.-85° C.

EXAMPLE 4 Thermus T-351 Growth and Caldolysin Production

Optimum production of Caldolysin was achieved when Thermus T-351 wasgrown on peptone media containing Allen's salts at peptoneconcentrations of 0.6% to 1%, (Cell division time˜2 hours).Concentrations of peptones greater than 1% inhibited production. ThermusT-351 grew poorly on salts/casein or salts/albumin media, and excretedlittle protease. However, yields of extracellular protease could beincreased significantly by addition of protein substrates to 0.6%peptone media. At 75° C., optimum yield of Caldolysin occurred within 18hours (19% inoculum, aeration=media vol./min.).

Yield: 0.12 PU/ml culture medium: where 1 PU=1 mg tyr released/min at75° C.; substrate=0.5% casein.

EXAMPLE 5 Immobilisation of Caldolysin to Glass Beads

Caldolysin was immobilised on non-porous glass beads by the silaneglutaraldehyde-coupling method described by Stolzenbach & Kaplan((1976). Methods in Enzymology 44, 926). 10 g of glass beads (Corningglass, 100 mesh) was washed in an excess of 5% HNO₃ at 100° C. for 30minutes. The acid-washed glass was filtered and rinsed, then added to a10% aqueous solution of γ-aminopropyl triethoxysilane (adjusted to pH3.5 with HNO₃). The suspension was incubated at 75° C. for approximatelythree hours to permit silanization to occur. After filtering, thesilanized glass was added to a 20 ml volume of 5% glutaraldehyde in0.01M, pH 7, phosphate buffer. This was reacted in vacuo for two hoursat room temperature, and finally washed exhaustively with distilledwater.

17 ml of a solution of Caldolysin (25 μg ml⁻¹) of known activity wasadded to the prepared ceramic substrate. The suspension was stirred atroom temperature for 18 hours to complete glutaraldehyde crosslinking.The immobilised enzyme was subsequently filtered, washed with 100 ml H₂O, 100 ml 1M NaCl, and a further 500 ml H₂ O. The filtrate and washingswere assayed by the Kunitz method.

The immobilised complex was assayed by a modification of the Kunitzmethod. 14 mg samples of the enzyme-bead complex were placed in reactiontubes, mixed with 2 ml of 0.5% casein substrate, and incubated at 75° C.with continual shaking. The proteolytic activities of the originalenzyme solution, the immobilised preparation, and the washings(non-immobilised enzyme) were calculated (Table 11).

                  TABLE 11                                                        ______________________________________                                        Activity of Glass-bead-immobilised CALDOLYSIN                                                       Enzyme                                                                        activity (PU)                                           ______________________________________                                        Total enzyme activity of original                                                                     25.4                                                  solution                                                                      Total enzyme activity not bound to                                                                    0.6                                                   glass beads                                                                   Total activity of ceramic-bound enzyme                                                                0.2                                                   Recovery of activity in immobilised state =                                                           1%                                                    ______________________________________                                    

It is concluded that Caldolysin was either inactivated during theattempt to cross-link it to the silanized glass, or was bound in such anorientation that steric hindrance prevented access of the proteinsubstrate to the catalytic site.

EXAMPLE 6 Immobilisation of Caldolysin to Sepharose 4B

Sepharose 4B (Pharmacia) was activated with cyanogen bromide asdescribed by Fujiwara & Tsuru ((1977) International Journal of Peptideand Protein Research, 9, 18). During activation, the Sepharosesuspension was maintained at 25° C., and at pH 10 to 11 by dropwiseaddition of 4N NaOH. The activated gel was washed and stored at 4° C.

15 ml of a Caldolsyin solution (25 μg ml⁻¹ in 0.1M CH₃ COONa, pH 7.2)was adjusted to pH 9.7 and added to 40 ml of settled activated Sepharose4B. The mixture was incubated at 4° C. for 72 hours. Subsequently, theCaldolysin-Sepharose complex was filtered and washed with distilledwater. Assay results for the free enzyme, immobilised enzyme and gelwashings are presented in Table 12.

                  TABLE 12                                                        ______________________________________                                        Activity of Sepharose 4B-immobilised CALDOLYSIN                                                     Enzyme                                                                        activity (PU)                                           ______________________________________                                        Total activity of free enzyme solution                                                                17.0                                                  Total activity not bound to Sepharose                                                                 0.7                                                   Total activity of Sepharose-bound enzyme                                                              12.0                                                  Recovery of activity in immobilised state =                                                           73%                                                   ______________________________________                                    

EXAMPLE 7 Immobilisation of Caldolysin to Carboxymethylcellulose

The Curtius azide method, first described by Michael & Ewers ((1949)Makromolekular Chemie 3, 200) modified by Mitz & Summaria ((1961) Nature189, 576) and detailed by Crook et al. ((1970) Methods in Enzymology 19,963) and Lilly ((1976) Methods in Enzymology 44, 46) was used toimmobilise Caldolysin to CM-cellulose. 5 g of CM-cellulose (Pharmacia)was treated with methanol in acid, hydrazine hydrochloride, and sodiumnitrite in acid, as described in the papers cited above.

To the activated cellulose was added 77 ml of Caldolysin (61.5 μg ml⁻¹in pH 9.2 buffer). The substrate-enzyme coupling reaction wasaccompanied by a decrease in pH, which was readjusted to 8.7 by additionof saturated sodium borate solution during the 60 minute duration ofreaction. The complex was subsequently washed with aliquots of distilledwater, NaCl, acetic acid, and sodium bicarbonate solutions. Theimmobilised complex and all solutions were assayed as previouslydescribed. Activity data are presented in Table 13.

                  TABLE 13                                                        ______________________________________                                        Activity of CALDOLYSIN immobilised to CM-cellulose                                                  Enzyme                                                                        activity (PU)                                           ______________________________________                                        Total activity of free enzyme                                                                         239                                                   solution                                                                      Total activity not bound to CM-                                                                       29                                                    cellulose (washings)                                                          Total activity of CM-cellulose-                                                                       66                                                    immobilised CALDOLYSIN                                                        Recovery of activity in immobilised state =                                                           31%                                                   ______________________________________                                    

EXAMPLE 8 Comparative data for free and immobilised Caldolysin

It has been shwon in examples 5 to 7 that the immobilisation ofCaldolysin to various insoluble substrates occurs with considerabledifferences in the recovery of active immobilised enzyme (i.e. 1% forglass beads, 31% for CM-cellulose, and 73% for Sepharose 4B). This maybe due to loss of activity by denaturation, or differences in inhibitiondue to the site of the enzyme-matrix covalent linkage.

The activity retained after immobilisation of Caldolysin to Sepharose(73%) was high when compared to other published data. In binding a rangeof proteases to Dowex MWA-1 anion exchange resin, Ohmiya et al. ((1978)Biotechnology and Bioengineering 20, 1), found activity yields rangingfrom 3% to 39%. Mason et al. ((1975) Biotechnology and Bioengeneering17, 1019) obtained activity yields of 41.4% and 57.7% on coupling B.subtilis neutral protease to glass by the azo- and glutaraldehydemethods, respectively.

A range of characteristics of the immobilised Caldolysin preparations,including thermostabilities, pH activity profiles, and Michaelis-Mentenkinetics, were compared with those of the free enzyme. Since theresidual activity of the glass-bead immobilised enzyme was extremelylow, no further study of this complex was carried out.

The thermostabilities of the immobilised Caldolysin preparations weredetermined at different temperatures and calcium concentrations. Volumesof immobilised enzyme were suspended in 0.1M Tris acetic acid buffer, pH8.1, containing known concentrations of calcium. The suspensions wereincubated at the desired temperature, and aliquots removed at intervalsfor assay after agitation of the suspension to ensure homogeneity.Immobilised apoenzyme suspensions were obtained by eluting the insolublecomplex (held in a Pharmacia K12 glass column) with 10 mm EDTA forseveral hours, and final washing with distilled water. (The term"apoenzyme" is subject to the conditions discussed previously: it ispossible that in the immobilised state, tightly bound calcium ions mightnot be removed by such treatment). Thermostability data is presented inTable 14.

                  TABLE 14                                                        ______________________________________                                         A comparison of the thermostabilities of free and immobilised                CALDOLYSIN                                                                                             Half-life                                                      Calcium                                                                              Ca.sup.2+                                                                             (minutes) at T° C.                            Enzyme status                                                                             status   (mM)    85   90      95                                  ______________________________________                                        Free        Holo     10       360 60       28                                 Sepharose-bound                                                                           Holo     10      1060 165     125                                 CM-cellulose-bound                                                                        Holo     10      --   110     --                                  Free        Apo      0       --   >6      --                                  Sepharose-bound                                                                           Apo      0       --   28      --                                  Free        Holo     0       --   approx. 15                                                                            --                                  Sepharose-bound                                                                           Holo     0       --   64      --                                  ______________________________________                                    

The immobilisation of Caldolysin on Sepharose results in an increase inthermostability of 3 to 4-fold over a number of different temperaturesand conditions, while a thermostability increase of approximately 2-foldresults from covalent linkage to CM-cellulose. The decrease in stabilityof the holo-enzyme Sepharose complex when incubated in a calcium-freebuffer suggests that the stabilisation by high calcium concentrations isas significant a factor in the immobilised state as in the free enzyme,while the decreased stability of the Sepharose-immobilised enzyme afterEDTA treatment ("apoenzyme") indicates that immobilisation does notprevent the removal of at least some of the calcium-conferredstabilisation.

A sample of Thermus T-351 has been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A.under number 31674.

EXAMPLE 9 Stability of Caldolysin at varying concentrations of Ca⁺⁺

The half-lives of Caldolysin in the presence of different concentrationsof calcium ions is presented in Table 15. The Caldolysin and ionsolutions were prepared as in Example 3(C) (3) above.

                  TABLE 15                                                        ______________________________________                                        Ca.sup.++  Concentration                                                                        Half life                                                   mM                min.                                                        ______________________________________                                        0                 <10                                                         0.1                15                                                         0.5                22                                                         1                  38                                                         5                 225                                                         10                ˜360                                                  50                600                                                         100               780                                                         500               780                                                         ______________________________________                                    

What is claimed is:
 1. A proteolytic enzyme derived from themicroorganism Thermus aquaticus (Variety T-351), ATCC No. 31,674, havingan isoelectric point of about pH 8.5, a molecular weight of20,000±3,000, stability at temperatures up to 75° C. at pH values from 4to 12 in the presence of divalent cations, and being destabilized byEDTA.
 2. An enzyme according to claim 1 in which said divalent cationsare calcium ions.
 3. An enzyme according to claim 1 or 2 immobilised ona suitable substrate.
 4. An immobilized enzyme according to claim 3wherein said substrate is an activated agarose affinity matrix.
 5. Animmobilized enzyme according to claim 3 wherein said substrate iscarboxymethyl-cellulose.
 6. A process for preparing an enzyme accordingto claim 1, which process comprises the steps of:(i) centrifuging aculture of Thermus aquaticus (Variety T-351), ATCC No. 31,674; (ii)subjecting the supernatant solution from said centrifugation to affinitychromatography on a suitable activated affinity chromatography gel; and(iii) eluting said enzyme from said gel with a suitable eluting agent.7. A process according to claim 6, which includes the preliminary stepof culturing said Thermus aquaticus (Variety T-351), ATCC No. 31,674 ina culture containing 0.1 to 0.3 w/v percent yeast extract, 0.1 to 0.3w/v percent trypticase extract, peptone, Allen's salts and at atemperature of 75° C. for up to 18 hours.
 8. A process according toclaim 6 or 7 wherein said centrifugation is continuous flowcentrifugation conducted at up to 30,000 g.
 9. A process according toany one of claims 6 or 7 wherein said supernatant solution from saidcentrifugation is passed through a suitable ion exchange gel andsubsequently subject to ultrafiltration prior to said affinitychromatography step.
 10. A process according to claim 9 wherein said ionexchange is conducted with a cationic ion exchanger comprising dextrancross-linked with epichlorohydrin and having sulphopropyl functionalgroups attached by ether linkages to glucose units of the dextranchains.
 11. A process according to claim 10 wherein said affinitychromatography gel is agarose activated with cyanogen bromide.
 12. Aprocess according to claim 11 wherein said ultrafiltration is conductedon a medium scale millipore ultrafiltration unit having a molecularweight 1000 nominal cut-off membrane and continued for a time sufficientto concentrate the solution ten times.
 13. A process according to claim12 wherein said solution subjected to affinity chromatography isadjusted to pH 8 prior to being so subjected.
 14. A process according toclaim 13 wherein said affinity gel is sequential reaction of agarosewith cyanogen bromide, trimethylene tetramine,carbobenzoxy-D-phenylalanine, and 1-ethyl-3-(3-dimethyl amino propyl)carbodiimide.
 15. A process of claim 14 wherein said affinity gel iseluted with a 0.1M acetic acid at pH 2.7 containing 10 mM Ca²⁺.
 16. Aprocess according to claim 15, which includes the additional stepsof:(a) subjecting said solution, after it has been subjected to saidaffinity chromatography to ultrafiltration to give an ultrafiltrationproduct; and (b) passing the ultrafiltration product of step (a) througha beaded dextran gel cross-linked with epichlorohydrin and having a beadsize from 10-40 mm diameter in a gel column at pH 8.1, the solutioncontaining 10 mM Ca²⁺ eluting buffer.